Mechanistic Insights into Cartilage Protection and Extracellular Matrix Remodeling: Transcriptome Analysis of Diclofenac Etalhyaluronate-Treated Knee Cartilage in Collagen-Induced Arthritis Rats and Cytokine-Stimulated Human Chondrocytes

Abstract

Background

Diclofenac etalhyaluronate (DF-HA, SI-613/ONO-5704) is a conjugate of hyaluronic acid (HA) and diclofenac (DF), and its intra-articular injection is widely used for the treatment of osteoarthritis in Japan. While novel mechanisms of cartilage protection by DF-HA have been identified, a comprehensive analysis of the biological responses unique to DF-HA has not yet been conducted.

Design

We used an RNA sequencing (RNA-seq) method to comprehensively analyze gene expression in the knee joint cartilage of arthritic rats and cytokine-stimulated chondrocytes. For the mechanistic analysis of DF-HA, genes that were downregulated or upregulated by DF-HA, HA, or DF were extracted. Pathway analysis was then performed on genes that specifically varied with DF-HA treatment.

Results

In the cartilage of rats with collagen-induced arthritis, treatment with DF-HA, but not DF or HA, suppressed the extracellular matrix (ECM) remodeling pathway and promoted the parathyroid hormone/parathyroid hormone-related peptide receptor-mediated pathway, which regulates chondrocyte differentiation and bone/cartilage development. In cytokine-stimulated chondrocytes, DF-HA similarly suppressed the ECM remodeling pathway; specifically, gene expression changes in IGFBP4, MMP10, MMP13, and TIMP1 were consistent with those observed in vivo.

Conclusion

RNA-seq analysis of cartilage in arthritic rats and cytokine-stimulated chondrocytes provided molecular mechanistic insights, indicating that DF-HA treatment induced cartilage protection through the suppression of ECM remodeling.

Keywords

diclofenac etalhyaluronate cartilage protection RNA sequencing extracellular matrix remodeling osteoarthritis gene expression collagen-induced arthritis

Introduction

Articular cartilage is important for smooth joint movement due to its shock-absorbing and low-friction properties.¹ Cartilage degeneration is a pathological condition commonly observed in patients with arthropathies, such as osteoarthritis (OA) and rheumatoid arthritis, and the causes of the condition are biological or mechanical factors exemplified with inflammation or excessive loading, respectively.² As cartilage degeneration progresses, joint pain is presumed to arise due to the exposure of subchondral bone, increased bone pressure, and secondary synovitis.³ Therefore, inhibiting the progression of cartilage degeneration is important in the treatment of the arthropathies, but there are currently no drugs available in clinical practice, which inhibit the cartilage damage progression.

Diclofenac etalhyaluronate (DF‑HA; product name, JOYCLU^®; development code, SI-613/ONO-5704) is known to reduce pain in OA patients in clinical trials.^4
-6 DF‑HA is a compound consisting of diclofenac (DF), a nonsteroidal anti-inflammatory drug, and hyaluronic acid (HA), which are covalently linked via a 2-aminoethanol linker. Gradually released DF from DF‑HA by hydrolysis of the linker contributes to its anti-inflammatory and analgesic effects.⁷ In addition, our preclinical study using collagen-induced arthritis (CIA) model rats and cytokine-stimulated chondrocytes have shown that DF‑HA inhibits cartilage degeneration partly by suppressing the production of matrix metalloproteinases, MMP3 and MMP13, in cartilage and synovium.⁸ The inhibitory effect on MMP3 production in the cartilage of the CIA model was unique to DF‑HA, and was not observed with DF or HA when used on their own. As another unique effect of DF-HA, Kisukeda et al.⁹ reported the induction of high-molecular-weight HA production from synovial cells by DF-HA.

Novel mechanisms of action of DF-HA on the chondroprotective effect have been increasingly revealed; however, no further comprehensive exploration of the mechanisms unique to DF-HA using methods, such as transcriptome analysis, has been conducted. There have been reports where comprehensive gene analysis using RNA sequencing (RNA-seq) has provided insights into the mechanisms of action of several drugs.^10,11 Thus, we analyzed and compared the effects of DF‑HA and its components (HA and DF) on transcriptome status in the knee cartilage of CIA rats and in cytokine-stimulated chondrocytes using RNA-seq to understand the unique mechanism of action of DF-HA.

Method

Test Compounds

DF-HA (DF: approximately 11.8% [w/w]) and HA (600-1,200 kDa) were manufactured by Seikagaku Corporation (Tokyo, Japan). DF was purchased from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). DF-HA and HA were dissolved in phosphate-buffered saline (PBS, Thermo Fisher Scientific Inc., Waltham, MA). DF was prepared in distilled water (Otsuka Pharmaceutical Factory Inc., Tokushima, Japan).

Animals

Nine-week-old female Dark Agouti rats were purchased from Japan SLC, Inc. (Shizuoka, Japan). The rats were maintained in specific pathogen-free conditions at a room temperature ranging from 20 to 26°C, humidity of approximately 50%, and a 12-hour light/dark cycle. The animal study protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Seikagaku Corporation. The animal studies were performed under an animal husbandry management system in an appropriate environment to ensure animal welfare.

Induction of Arthritis Model and Administration of Test Compounds

The bovine collagen type II solution (Collagen Research Center, Tokyo, Japan) was emulsified by mixing it with an equal volume of incomplete Freund’s adjuvant (Becton Biosciences, Franklin Lakes, NJ). Under isoflurane inhalation anesthesia, rats were immunized by intracutaneous injection of the emulsion (0.05 mg/0.1 mL/site) at four sites on the tail root to induce autoimmune arthritis. The normal control rats underwent the same procedures but received intracutaneous injections of physiological saline. Two weeks after immunization, 50 μL of PBS, DF-HA (0.5 mg), or HA (0.5 mg) was administered into the articular cavity of the knees of both hindlimbs under isoflurane anesthesia. DF (2 mg/kg) was orally administered once a day from 2 to 3 weeks after immunization.

Histological Analysis

The rats were exsanguinated under isoflurane anesthesia, and the proximal portion of the tibia at the knee joints of left hindlimbs was collected at 2 (before the administration of test compounds) and 3 weeks after immunization. Twelve rats per group were evaluated. The tibial plateau of knee joints was fixed in 10% neutral-buffered formalin (Fujifilm Wako Pure Chemical Corporation) and decalcified in formic acid formalin (Thermo Fisher Scientific Inc.). The lateral tibia was sectioned longitudinally and embedded in paraffin. The sections were stained with safranin O (Waldeck GmbH & Co. KG, Münster, Germany) and fast green (Nacalai Tesque, Inc., Kyoto, Japan), and the severity of cartilage degeneration was scored according to a slightly modified Mankin scoring system. Briefly, surface structure was scored from 0 to 5; 0, normal structure; 1, surface irregularities; 2, pannus and surface irregularities; 3, clefts to transitional zone; 4, clefts to calcified zone; and 5, complete disorganization. Cellularity was scored from 0 to 3; 0, normal; 1, diffuse hypercellularity; 2, cloning; and 3, hypocellularity. Safranin O staining intensity was scored from 0 to 4; 0, normal; 1, slight reduction; 2, moderate reduction; 3, severe reduction; and 4, no dye noted. Tidemark was scored 0 or 1; 0, normal and 1, destroyed. The effects of the test materials were evaluated in the anterior one third of the tibial cartilage, where degeneration due to model progression was evident. Scoring was performed in a blinded manner. The histological scores of each group, except for the normal group, were statistically analyzed using the Steel-Dwass test (SAS software package in EXSUS, CAC Croit Corporation, Tokyo, Japan). Differences were considered statistically significant at P < 0.05.

RNA Extraction from Rat Knee Joint Tissue

Tissue collection was performed at 2 (before drug administration) and 3 weeks after immunization. Eight rats per group were evaluated. The cartilage of the right knee joints was collected and rapidly frozen in liquid nitrogen. Cartilage was homogenized in TRIzol reagent (Thermo Fisher Scientific Inc.). Total RNA was extracted using the PureLink RNA Mini Kit (Thermo Fisher Scientific Inc.) according to the manufacturer’s instructions, and RNA concentrations were measured using a microvolume spectrophotometer (NanoDrop 2000c, Thermo Fisher Scientific Inc.). The RNA quality parameter of the cartilage was evaluated with the 4200 TapeStation system (Agilent Technologies, Inc., Santa Clara, CA, USA). The RNA integrity number (RIN) had a mean of 8.76, a median of 8.9, a mode of 8.9, a minimum of 7.2, and a maximum of 9.5.

Culture of Chondrocytes

The human chondrocytes (Cell Applications, Inc., CA, USA) were cultured in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) containing 10% fetal bovine serum (FBS) at 37°C in 5% CO₂. Cells at passage 1 were seeded in six-well plates at a density of 3 × 10⁵ cells per well and cultured for 1 day. The medium was replaced with 2 mL of DMEM/F-12 containing 10% FBS and the corresponding materials: DF-HA (2 mg/mL), HA (2 mg/mL), or DF (3 µg/mL). After 30 minutes, cells were stimulated with interleukin-1 beta (IL-1β) (0.1 ng/mL) for 16 hours. Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen N.V., Venlo, The Netherlands) according to the manufacturer’s protocol, and RNA concentrations were measured using a microvolume spectrophotometer. The RIN had a mean of 9.80, a median of 9.8, a mode of 9.9, a minimum of 9.4, and a maximum of 9.9.

Next-Generation Sequencing

Total RNA (200 ng) was used to prepare libraries with the TruSeq Stranded mRNA Library Prep Kit (Illumina, Inc., San Diego, CA, USA). Prepared libraries (100 bp, paired end) were sequenced on NextSeq 2000 with 100 bp pair-end reads (Illumina, Inc.). The fastq files were generated using Illumina DRAGEN BCL Convert (v 3.9.3).

RNA-seq Data Processing

Quality checks of the sequence data were performed using FastQC (v 0.11.9). Each read in the fastq files was mapped to the genome file using RSEM (v 1.3.1). The quality of the mapping results was checked using MultiQC (v 1.8). Subsequently, the number of reads mapped to exon regions was calculated for each gene using featureCounts. Normalization of the read counts obtained was performed using the edgeR package in R, and counts per million (CPM) were calculated.

Differential Expression and Pathway Analysis

For comparisons between groups, count data were analyzed using the DESeq2 package (v 1.32.2) in R. The Wald test was used for statistical testing. Pathway analysis was performed using MetaCore^TM (Clarivate Analytics). MetaCore^TM is a web-based systems biology analysis tool used for RNA-seq analysis and other applications. It utilizes manually curated pathway maps for pathway detection. Significant pathways were detected using gene sets with|log2 fold change| > 0.59 and Benjamini-Hochberg (BH)-adjusted P value < 0.05 obtained from two-group comparisons, as well as gene sets extracted using Venn diagrams. The threshold of |log2 fold change| > 0.59 corresponds to a 1.5-fold change, a commonly used cut-off for biological significance in transcriptome studies. Heatmaps were generated using the pheatmap package (v 1.0.12) with scaled log2(CPM+2) values of genes included in the identified pathways.

Correlation Analysis Between Gene Expression and Pathology Scores

Correlation analysis between gene expression and pathology scores was conducted using Spearman’s rank correlation coefficient. Extracellular matrix (ECM) remodeling-related genes were selected, and their correlation with pathology scores was evaluated across all groups. Statistical significance was determined with a threshold of P < 0.05. A smoothing curve was fitted using locally estimated scatterplot smoothing (LOESS) to visualize the relationship between gene expression and pathology scores.

Results

Increased Cartilage Degeneration and Protective Effect of DF-HA Treatment by Histopathological Examination

To compare the chondroprotective effects of DF-HA and its components on the disease model, histopathological evaluations were conducted at 2 and 3 weeks post-CIA-induction. Supplementary Figure S1A shows the histopathological images of normal and CIA model rats at 2 weeks post-CIA-induction. Cartilage degeneration began 2 weeks after CIA-induction, and further deterioration was expected to follow; therefore, the compound administration started at this time point to examine the chondroprotective effect of the compounds at 3 weeks post CIA-induction ( Fig. 1A ). As shown in Figure 1B , the Mankin score was significantly reduced in the DF-HA-treated group compared with the arthritis-induced model (Safranin O staining intensity, P = 0.0130; total score, P = 0.0048). The DF group also showed a significant Mankin score reduction (tide mark, P = 0.0288; total score, P = 0.0400). However, no significant effect was observed in the HA group.

Figure 1.

Superior cartilage tissue protective effect of the DF-HA compared to other drugs. (A) Representative microscopic images of the tibial cartilage of a knee joint from each treatment group. Sections of tibial cartilage were stained with safranin O and fast green. The upper images present an overview of the cartilage. The lower images represent the squared area of the corresponding upper image at high magnification. Scale bar = 500 μm (upper images) and 50 μm (lower images). (B) Tibial sections were scored for signs of cartilage degeneration based on the Mankin scoring system. Values represent the mean ± standard error (see the “Materials and Methods” section for details).

Identification of Differentially Expressed Genes in the CIA Model and Pathways That Induce Cartilage Degeneration

To further analyze the molecular mechanisms underlying the disease pathology, we examined differentially expressed genes (DEGs) between normal and CIA model rats ( Fig. 2A ) at the 2 and 3 weeks post-CIA-induction ( Fig. 2B and Supplementary Fig. S1B, see also Supplementary Table S1 for all the results). For the DEGs identified between normal and CIA model rats, BH-adjusted P values at 2 weeks ranged from 4.7 × 10⁻²²³ to 1.00 (median = 0.01, mean = 0.18), with log2 fold changes ranging from −8.85 to 5.49 (median = 0.031, mean = 0.061). At 3 weeks, BH-adjusted P values ranged from 9.8 × 10⁻²⁹² to 1.00 (median = 0.003, mean = 0.16), with log2 fold changes ranging from −6.80 to 7.05 (median = −0.016, mean = 0.071). Pathway analysis using MetaCore^TM was also performed to explore the pathways activated in each group. MetaCore^TM is a manually curated database that collects molecular interactions from the literature, allowing exploration of the biological pathways associated with the target gene sets. The pathway analysis results from MetaCore^TM’s pathway map indicated that pathways related to Wnt and Hedgehog signaling, as well as cartilage and bone formation pathways, were enriched among the downregulated genes in CIA model rats ( Fig. 2C and D at 3 weeks and Supplementary Fig. S1C at 2 weeks after CIA-induction, see also Supplementary Table S2). Inflammation-related pathways were significantly enriched among the upregulated genes in both at 2 and 3 weeks (Supplementary Fig. S2, see also Supplementary Table S2).

Figure 2.

Identification of differentially expressed genes in the CIA model and pathways that induce cartilage degeneration. (A) Diagram of the RNA-seq analysis process applied to cartilage tissues from normal and CIA model rats. (B) Volcano plot illustrating upregulated and downregulated genes in cartilage tissue at 3 weeks post-CIA induction. (C) Pathway analysis results indicating significant enrichment of Development Induction of chondrogenesis and ossification by NOTCH signaling pathways in CIA model. Red bars represent DEGs within the pathways. (D) Heatmap showing DEGs involved in Wnt and Hedgehog signaling pathways in normal versus CIA model rats.

Suppression of ECM Remodeling Was Observed With the DF-HA Treatment, but Not With HA or DF in Cartilage Tissues

To elucidate the mechanisms underlying the cartilage-protective effects specific to DF-HA, RNA-seq was performed on cartilage tissue from each treatment group ( Fig. 3A and Supplementary Table S3). For the DF-HA group, BH-adjusted P values ranged from 4.7 × 10⁻²²³ to 1.00 (median = 0.010, mean = 0.18), with log2 fold changes ranging from −8.85 to 5.49 (median = 0.032, mean = 0.061). For the HA group, BH-adjusted P values were all 1.00, with log2 fold changes ranging from −4.06 to 2.99 (median = 0.0002, mean = −0.021). For the DF group, BH-adjusted P values ranged from 2.4 × 10⁻⁴ to 1.00 (median = 0.72, mean = 0.68), with log2 fold changes ranging from −4.59 to 4.31 (median = −0.008, mean = −0.022). We identified DEGs (|log2 fold change| > 0.59 and BH-adjusted P < 0.05) between the DF-HA-treated group and untreated groups ( Fig. 3B ), as well as those between the DF- or HA-treated group and untreated groups (Supplementary Table S3). The Venn diagrams show comparison among CIA-upregulated DEGs and DF-HA-, DF-, and HA-downregulated DEGs ( Fig. 3C ) and that among CIA-downregulated DEGs and DF-HA-, DF-, and HA-upregulated DEGs ( Fig. 3D ). Next, pathway analysis was performed on the DEGs specifically upregulated or downregulated by DF-HA treatment (see Supplementary Table S4 for all the results). DF-HA treatment resulted in the enrichment of ECM remodeling pathway among the downregulated DEGs and the parathyroid hormone receptor 1 (PTHR1) bone and cartilage development pathway among the upregulated DEGs ( Fig. 3E and F ). The DEGs involved in ECM remodeling pathway, such as IGFBP4, MMP10, MMP13, MMP14, and TIMP1, and PTHR1 bone and cartilage development pathway, such as Adcy2, Col2a1, Ihh, and Ptch1, were differentially expressed in the disease state and were specifically suppressed or enhanced in the DF-HA-treated group, as visualized in the heatmaps ( Fig. 3G and H ). Notably, the expression of ECM remodeling-related DEGs showed a significant correlation with pathology scores across all comparisons (Supplementary Fig. S3), emphasizing the role of ECM in disease pathology and treatment response.

Figure 3.

Suppression of ECM remodeling as a result of DF-HA treatment. (A) Diagram of the RNA-seq analysis process applied to cartilage tissue for each treatment group. (B) Volcano plot comparing gene expressions in DF-HA treated CIA rats versus untreated CIA rats. (C, D) Venn diagrams comparing CIA-upregulated DEGs with downregulated DEGs by the component treatments (C) and CIA-downregulated DEGs with upregulated DEGs by the component treatments (D). For the Venn diagram analysis, DEGs were defined as genes with BH adjusted P value < 0.05 and |log2 fold change| > 0.59.(E, F) Pathway analysis showing the impact of DF HA treatment on transcriptome alteration in the CIA model cartilage tissue: the suppression of ECM remodeling pathways (E) and enhancement of PTHR1 bone and cartilage development pathways (F). Red bars indicate DEGs. (G, H) Heatmap showing expression levels of DEGs involved in ECM remodeling (G) and PTHR1 pathways (H) suppressed or enhanced by DF-HA treatment in cartilage.

In Vitro Tests With Human Chondrocytes Recapitulated the Suppression of ECM Remodeling by DF-HA Treatment

To validate the in vivo findings, we conducted in vitro analysis using human chondrocytes. Test compounds were added 24 hours after cell seeding, followed by IL-1β stimulation 30 minutes later. RNA was extracted from the cells 16.5 hours after compound addition, and RNA-seq was performed ( Fig. 4A ). In the IL-1β-stimulated group, 1,608 DEGs were significantly upregulated, and 1,334 DEGs were downregulated. With the addition of DF-HA, 1,529 DEGs were upregulated, and 1,492 DEGs were downregulated ( Figs. 4B , C and Supplementary Table S5). The enrichment of the ECM remodeling pathway, which was specifically suppressed by DF-HA in the in vivo study, was also observed among the DEGs downregulated by DF-HA in vitro. Expression analysis of genes included in the ECM remodeling pathway revealed that IGFBP4, LAMA1, MMP10, MMP13, and TIMP1 were upregulated by IL-1β stimulation and downregulated by DF-HA treatment ( Fig. 4D ).

Figure 4.

In vitro transcriptome analysis with human chondrocytes recapitulated the suppression of ECM remodeling by DF-HA treatment. (A) Diagram of in vitro experiments using human cartilage-derived chondrocytes stimulated with IL-1β and treated with compounds (n = 3 per group). (B, C) Volcano plot illustrating upregulated and downregulated genes under IL-1β stimulation versus non-stimulated (B) and DF-HA treated versus non-treated groups (C). (D) Heatmap showing ECM remodeling-related genes which were downregulated in DF-HA treated CIA-induced rats in Fig. 4 in IL-1β stimulated human chondrocytes treated with test compounds.

Discussion

In OA, joint inflammation is relatively mild and has sometimes been seen as “non-inflammatory.” However, recent preclinical and clinical evidence strongly supports the view that inflammation plays a central role in OA pathogenesis.¹² Therefore, in this study, we used the CIA model, characterized by severe synovitis as well as cartilage and bone destruction,¹³ to investigate cartilage degeneration associated with inflammatory arthritis. Knee cartilage degeneration induced by CIA was suppressed by the administration of DF‑HA, replicating the results of our previous study.⁸ Therefore, we conducted a comprehensive analysis of gene expression changes associated with the pathological condition in which DF-HA is effective in protecting cartilage, using RNA-seq.

Among the cartilage and bone formation pathways, Sox9, Acan, and Col2a1 are important for the phenotype of hyaline cartilage chondrocytes.¹⁴ In particular, Sox9 is an essential transcription factor for chondrocyte specification and differentiation, and its expression is decreased by chondrocyte hypertrophy, which is one of the degenerative changes in cartilage.¹⁵ Among inflammation-related genes, the upregulation of Cebpb,¹⁶ Rela,¹⁷ Batf,¹⁸ and Irf8¹⁹ is considered to be associated with cartilage degeneration. Therefore, it was reasonable that the changes in gene expression of cartilage and bone formation pathways and inflammation-related pathways were associated with cartilage degeneration in the CIA model. Conversely, upregulation of Wnt signaling and Hedgehog signaling has been reported to contribute to cartilage degeneration in many OA studies;^20
-22 however, these pathways were downregulated in the CIA model. As the CIA model represents inflammatory arthritis, this downregulation is likely caused by inflammatory stimuli, as reported by Cici et al.²³ and Thompson et al.²⁴ The impact of this downregulation on cartilage degeneration remains unclear, and the CIA model shows limited similarity to OA pathology with respect to the regulation of these pathways. Based on these findings, the downregulation of cartilage and bone formation pathways and the upregulation of inflammation-related pathways were considered to be the main drivers of cartilage degeneration in this model.

DF-HA showed inhibitory effects on the ECM remodeling pathway; specifically, changes in IGFBP4, MMP10, MMP13, and TIMP1 were consistent between in vivo and in vitro. IGFBP4 is a secretory protein known to be increased in the cartilage²⁵ and synovial fluid²⁶ of patients with OA. IGFBP4 is an inhibitor of IGF-1;²⁷ IGF-1 is a protein thought to promote damage repair by normalizing cartilage metabolism,²⁸ and IGFBP4 may be involved in promoting cartilage degeneration through its inhibition. MMP10 and MMP13 are metalloproteinases characteristically expressed by hypertrophic chondrocytes.²⁹ They degrade collagen and proteoglycan,³⁰ which are components of cartilage ECM, and are involved in cartilage degeneration.^31,32 TIMP1 is an inhibitor of metalloproteinases, and its concentration in synovial fluid increases in OA.³³ The downregulation of TIMP1 by DF-HA may appear to counteract cartilage protection; however, in cartilage where metalloproteinase expression is suppressed by DF-HA, the demand for TIMP1-mediated regulation of metalloproteinase activity is likely reduced. This hypothesis is supported by evidence of synchronized TIMP1 and MMP3 expression in joint tissues.^34,35 In other words, in cartilage with decreased metalloproteinase expression, TIMP1 downregulation by DF-HA is thought to have little or no effect on cartilage degeneration. Taken together, the involvement of MMP13, consistent with our previous findings,⁸ and IGFBP4 and MMP10, newly identified in this study, appears reasonable given the functional roles of these molecules in the chondroprotective effects of DF-HA. However, the mechanism underlying DF-HA-mediated transcriptional regulation remains unclear. To clarify this, future studies should include detailed analyses, such as identifying molecules that directly interact with DF-HA.

This study has several limitations. One is that our analyses are based solely on changes in transcript levels and do not encompass the potential functional changes that may arise from protein translation or post-translational modifications. The extent to which the target pathways of DF‑HA identified in this study contribute to the phenotype after these processes is unclear. Verifying the regulatory effects of DF-HA on the ECM remodeling pathway at the protein level would be useful to further strengthen the discussion of this study. Specifically, future studies should analyze the effects of DF-HA on the production of IGFBP4, MMP10, and MMP13 in the articular cartilage of the CIA model using enzyme-linked immunosorbent assays, western blotting, and immunohistochemistry. Another important point is the need to evaluate the effects of DF-HA in animal models, focusing on non-inflammatory factors such as mechanical stress. This is because the present study focused on cartilage degeneration caused by inflammation using a CIA model and cytokine-stimulated chondrocytes. To comprehensively understand the effects of DF-HA on OA, it is necessary to also investigate its effects in both spontaneous and surgically induced OA models. Despite these limitations, our findings provide novel insights, as they highlight the cartilage-protective effects of DF-HA through inhibition of ECM remodeling, suggesting a unique therapeutic potential for DF-HA in cartilage protection during cartilage degeneration.

While the ECM serves as a scaffold to maintain tissue integrity and elasticity, the ECM is also known to control development through dynamic structural changes called remodeling in some tissues such as the intestine, lungs, mammary glands, and salivary glands.³⁶ On the contrary, dysregulated ECM remodeling is associated with disease states; for example, abnormal accumulation of ECM is implicated in the development and progression of diseases such as cancer and fibrosis, while excessive degradation of ECM is thought to be involved in OA. The findings obtained in this study contribute to the elucidation of the pathophysiology of cartilage degeneration and the mechanisms of action of DF‑HA. Moreover, the findings suggest that DF-HA may exert long-term benefits not only through analgesic effects but also via disease modification mediated by cartilage protection in OA. Although several compounds, including the broad-spectrum metalloproteinase inhibitor PG-116800, ADAMTS-5 inhibitors GLPG1972 and M6495, and cathepsin K inhibitor MIV-711, have been developed as disease-modifying OA drugs (DMOADs) targeting ECM degradation, challenges related to their efficacy and safety have hindered their application in clinical practice.³⁷ Given that DF-HA is already clinically available, the current preclinical findings motivate clinical validation studies regarding its disease-modifying effects, which are expected to advance the optimization of treatment protocols, including patient selection and dosing duration.

Conclusion

RNA-seq analysis of the cartilage from the CIA model and cytokine-stimulated chondrocytes revealed that DF‑HA-mediated cartilage protection involves suppression of ECM remodeling. Further studies are needed to elucidate the mechanism of action of DF‑HA at the protein level and to verify that the ECM remodeling suppressing effect of DF‑HA contributes to cartilage protection in a clinical setting.

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Footnotes

Acknowledgements

Completed graphics were created with .

ORCID iDs

Akihiro Kitani

Shuhei Takada

Shohei Nozaki

Kazuhiro Kojima

Jun Takeuchi

Keiji Yoshioka

Ethical Considerations

Animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Seikagaku Corporation and were performed under an animal husbandry management system in an appropriate environment with consideration given animal protection and welfare (approval number 78-010).

Consent for Participate

Not applicable.

Consent for Publication

Not applicable.

Author Contributions

A.Ki. contributed to the conception, design, data interpretation, data analysis, drafting, and revision of the manuscript. S.T. contributed to the conception and design of the study, interpretation of the data, and drafting and critical revision of the manuscript. S.N., K.K., K.T., and T.H. contributed to the acquisition, analysis, and interpretation of the data, and drafting of the manuscript. J.T., A.Ka., T.F., and K.Y. contributed to the conception and design of the study, and critical revision of the manuscript. All authors have given the approval of the version to be published.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Research was conducted as part of ONO Pharmaceutical Co. Ltd and Seikagaku Corporation’s research activities and was not externally funded.

Declaration of Conflicting Interests

The authors declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: A.Ki., S.N., T.H., A.Ka., and T.F. are employees of Ono Pharmaceutical Co., Ltd. and S.T., K.K., K.T., J.T., and K.Y. are employees of Seikagaku Corporation.

Data Availability

The RNA-seq data used in this study are available at the National Bioscience Database Center (NBDC) with the accession code of PRJDB19919.

Supplemental Material

Supplementary material for this article is available on the Cartilage website at .

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Supplementary Material

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